For producing a product of interest such as in particular a protein, recombinant techniques are widely applied. The product of interest is expressed in a suitable host cell and the expressed product is obtained e.g. from the cells and/or the cell culture medium.
The basic idea of fermentation is to maintain cells under optimal conditions for a period of time in different scales in order to produce high amounts of the desired product. Fermentation is common for procaryotes (e.g. Escherichia coli), yeast (e.g. Pichia pastoris) and mammalian cells (e.g. rodent cell lines such as Chinese hamster ovary cells (CHO), mouse myeloma cells (NSO; SP2/0) or human cell lines). Beside an optimized culture media, a well controlled fermentation process is the basis for a production process in mammalian cell culture. Depending on the production cell line, cells are either grown in suspension culture or on a carrier matrix for anchorage-dependent cell culture. Small scale cell culture is performed using a relative low level of instrumentation. For suspension culture roller bottles and spinner flasks are e.g. suitable. Small scale cell culture is usually operated in a humidified carbon dioxide incubator. As gas transfer in a carbon dioxide incubator is based upon passive diffusion, gas transfer limitations can occur. The technique of fermentation for the production of pharmaceutical products is covered extensively in the literature as numerous reviews are available e.g. [Andersen and Reilly 2004], [Shukla and Thömmes 2010], [Marks 2003] and [Morrow 2007]. Regarding the operation of cell culture fermentation, there are four different basic strategies for bioreactors, which have been described in the literature: batch, fed-batch, continuous fermentation without cell retention and continuous fermentation with cell retention (perfusion). As perfusion does include a cell retention, the only way of removing cells during cultivation is the usage of a bleed (removal of cell suspension).
Fermentation parameters are characteristic for a production process. Changing those parameters may increase or decrease the product yield. Inside the bioreactor temperature, dissolved oxygen and pH-value are basic parameters to be controlled. Strategies for optimization base upon adjustment of those parameters. Some parameters, which are suitable for optimization include but are not limited to the temperature used during fermentation, the used oxygen level, the cell retention and the composition of the used media.
Furthermore, also the chosen aeration/sparging can be an important feature for the culturing process. Cells growing in cell culture require oxygen for efficient growth. In small cultivation volumes the amount of oxygen that reaches the cells by diffusion via the surface of the cultivation medium is generally sufficient. However as the cultivation volume increases, the specific addition of oxygen becomes necessary. This is normally achieved by sparging, so that bubbles of the gas or gases to be supplied to the cell culture are introduced. When larger vessel sizes are required, homogeneous gas supply becomes more and more of a concern. However the gas supply cannot be arbitrarily increased because most mammalian cells tend to be sensitive to shear forces created by the bubbles. Said bubbles carry attached cells to the surface, where the bubbles rupture under formation of high hydrodynamic stress, thereby killing the attached cells. These lethal effects can reduce the cell viability and hence the productivity of a cell culture. Bubble-free gas supply systems (e.g. membranes) that may prove useful in smaller cultures are not practical for use in larger cultures during scale up. Reasons for this are for example the high costs associated with the use of membranes or technical limitations in the large vessels.
Furthermore, in order to achieve a homogenous cell culture, the suspension is usually mixed by agitation. This can be achieved e.g. by impeller agitation and gas sparging. For agitation usually a stirrer is used. However there are also limitations to agitation. Cells usually tend to be sensitive to the shear forces induced by agitation, for example stirring. Therefore, the potential for ensuring homogeneity by agitation is limited.
Regarding mixing, there are two specific problems: (i) the addition of base solution and feed solution and (ii) carbon dioxide accumulation. Concentration variations of substrate levels but also oxygen gradients can occur. They are more common in large vessels than in small vessels. Uneven distribution increases with vessel size and becomes more and more critical. The main problem of mixing is the trade-off between inhomogeneities and shear forces. To ensure sufficient mixing and non-damaging shear stress is an intriguing challenge. Both mixing and shear stress can lead to cell death by different means. Insufficient mixing can e.g. lead to clumping or oxygen limitation while shear stress can lyse cells.
Hydrodynamic stress based on shear forces is a major cell culture issue. Generally, damage by shear forces is to a very high extent cell line dependent. Additionally, as peak cell densities of fed-batch or perfusion processes increase due to process and medium optimisation there is an additional demand for mixing as the viscosity increases. Hydrodynamic stress in a stirred tank bioreactor is a non-homogeneous phenomenon. In impeller regions, which account for only about 10% of total volume, up to 70% of energy is dissipated. Consequently, also shear forces are much higher in those regions close to the impeller which may cause lethal or non-lethal damaging effects on the cells. Not only can hydrodynamic stress lyse cells; it may also influence cells on a sublytic level, which is currently not well understood.
These factors contribute to the fact that culturing conditions that might be suitable for small-scale cultures cannot be easily transferred to large scale cultures. The outcome of protein production in large-scale cultures is often considerably different from that of small-scale cultures. During large scale production, very often the productivity and/or the quality of the produced protein (e.g. the glycosylation structures in case the glycoprotein is recombinantly produced) is decreased. All together the scale up remains a major issue in mammalian cell culture. Although a lot of literature is available for scale up procedures regarding scale up concepts and considerations, scale up remains a challenge and measures suitable for one specific cell line, can very often not be transferred to a different cell line.
When producing a glycosylated product of interest, such as e.g. an antibody that is to be used in therapy, it is desirous to obtain a “human” or “humanised” glycosylation structure. Several techniques and host cells are available for that purpose. Immortilized human blood cells and cell lines derived therefrom were found suitable for recombinantly producing glycosylated products having a human glycosylation pattern. Respective cell lines are e.g. described in WO 2008/028686. Product glycosylation is a complex post-translational modification which may be affected by a lot of different parameters. These parameters can be of physical, chemical or thermodynamic nature. As these parameters can be affected during fermentation, it is very important to develop a fermentation process that allows to produce a product with a constant, i.e. homogenous glycosylation pattern. There are several reports to be found in the literature. It is e.g. reported that shear force, glucose availability, oxygen saturation, pH, temperature and other process conditions may affect glycosylation [Senger and Karim 2003] [Godoy-Silva et al. 2009] [Tachibana et al. 1994] [Kunkel et al. 1998] [Müthing et al. 2003] [Ahn et al. 2008] [Lipscomb et al. 2005].
It is an object of the present invention to provide a method for culturing cells, in particular immortalized human blood cells, which allows to produce a product of interest with acceptable yield and good quality also when using different fermentation volumes (scales).